In a normal human heart, cardiac contraction is initiated by the spontaneous excitation of the sinoatrial (“SA”) node that is located in the right atrium. The electrical current generated by the SA node travels to the atrioventricular (“AV”) node where it is then transmitted to the bundle of His and Purkinje network, which branches in many directions to facilitate coordinated contraction of the left and right ventricles.
The cellular basis for the aforementioned electrical impulse is the action potential (AP). The AP is conventionally divided into five phases (phases 0-4) in which each phase is defined by the cellular membrane potential and the activity of potassium, sodium, chloride, and calcium ion channel proteins that affect that potential. These channels, embedded in cell membranes, allow for electrical impulses to occur as they permit charged ions to rush through them. Propagation of electrical activity from an individual cardiac cell to surrounding cardiac tissue takes place through gap junctions, small pore-like structures that connect cardiac muscle cells to each other. The role of ion channels in cardiac electrical conduction is analogous to electrical conduction in other tissues such as skeletal muscle.
Some channels or gates have their own “non-provoked” rhythmic excitation also known as automaticity. The generation of cardiac automaticity is based on a complex interplay between at least four different channels of cationic (positive ion) nature: T- and L-type calcium channels, a cation channel named If, and potassium channels. The If channel has been termed the pacemaker channel. If channels have unique properties such as: 1) If channels open upon membrane hyperpolarization; 2) If channels allow for mixed cation current (Na+ and K+); 3) cyclic AMP (cAMP-cyclic adenosine monophosphate which serves as an intracellular messenger molecule) binds to the cytoplasmic site of the channel thereby accelerating its activation kinetics and shifting the voltage dependence of the cell to more positive voltages; and lastly 4) If channels are susceptible to blockade by extracellular Cs+ (cesium ion). The genes responsible for the If channel currents have recently been identified and belong to the HCN (hyperpolarization-activated cyclic nucleotide-gated) family. Four different isoforms have been identified in vertebrates (HCN1, HCN2, HCN3 and HCN4) and all except HCN3 have been found in the heart. HCN3 is specifically expressed in neurons.
HCN channels directly interact with intracellular cAMP so that an increase in cAMP levels results in increased If and more positive activation potentials. This increase thereby accelerates the heart rate (HR) in response to sympathetic stimulation. In contrast, muscarinic stimulation slows the heart rate in part due to a decrease in cAMP levels and a resulting reduction of If and more negative activation potentials. Ludwig, A. et al.; “Two pacemaker channels from human heart with profoundly different activation kinetics.” EMBO J. (1999) 18 (9):2323-2329. The importance of the HCN genes in regulating heart rate has recently been shown in a patient who suffered from mutation in his HCN4 gene. This mutation consisted of a complete deletion of the C-terminus of the gene which included the cAMP binding domain. This patient suffered from symptomatic bradycardia and an electronic pacemaker needed to implanted. These mutations were recreated in vitro experiments, and the mutated channel was expressed in a cell line. The mutated HCN4 channel was completely inresponsive to cAMP. See, J Clin Invest. 2003 May:111(10):1537-45.
HCN1 is primarily expressed in the brain and shows little dependence on cAMP. HCN1 is also expressed in the rabbit SA node and displays properties of brain h-channels in that it has a short AP. HCN2 and HCN4 are predominantly expressed in the heart, as well as in the brain, and produce currents similar to If. HCN1 is the fastest activating channel (25-300 ms), followed by HCN2 and HCN3 (180-500 ms), and lastly HCN4 (a few hundred ms to seconds). All four subunits induce pacemaker current similar to If if the units are expressed in heterologous expression systems. In addition, the four isoforms can interact with one another to form tetramers (couplings whereby the two isomers join to create a functionally different structure). The heteromerization of the isoforms changes pacemaker electrophysiology via altered activation kinetics (e.g., allows for modulation (increase or decrease) of heart rate). (Much B et al. J of Biol Chem; 44 (31): 43781-43786). While the exact stoichiometry of the heteromerized HCN channels has not been described yet, it is considered that these channels may form heteromers with a 3:1 ratio, but ratios of 1:1 or 1:3 are also possible as the HCN channels are known to form tetramers. In related rod photoreceptor cyclic nucleotide-gated channels, an asymmetrical stoichiometry of the two subunits present in the tetramers of 3:1 was determined. Zhong H et al. Nature 2002; 420: 193-196. Weitz D et al. Neuron 2002; 36: 881-889. Zheng J et al. Neuron 2002; 36: 891-896.
To avoid misunderstandings due to different naming of the same proteins, isoform nomenclature for the mouse brain is as follows: HCN1 corresponds to HAC2 (mBCNG-1), HCN2 corresponds to HAC1 (mBCNG-2) and HCN3 corresponds to HAC3 (mBCNG4).
In certain diseased states, the heart's ability to pace properly is compromised. For example, failure of SA nodal automaticity, resulting in an insufficient number of electrical impulses emanating from the SA node, is the most common cause of bradyarrhythmias (heart rhythm that is too slow). If slowing is enough so that the resultant heart rate is insufficient to meet the body's demand, symptoms result. Symptomatic bradycardia originating from the sinus node is part of a clinical syndrome characterized by brady- and tachyarrhythmias originating from a diseased sinus node, commonly referred to as sick sinus syndrome. Clinically, sick sinus syndrome is a very common problem and accounts for approximately 70% of all pacemaker implants in the general population. Other bradyarrhythmic disease states due to slowed or absent impulse propagation include the various degrees of AV block (e.g. 1st, 2nd, or 3rd). Tachyarrhythmias (heart rhythm that is too fast) and fibrillation are also a concern. These conditions present major problems ranging from cost of treatment to diminished quality of life and even death.
Currently, bradyarrhythmias are most commonly treated by the implantation of (exogenously driven) electronic pacemaker. While improving the lives of many patients, implantable pacemakers have a limited lifetime and consequently may expose a patient to multiple surgeries to replace the implantable pacemaker. Biological methods of influencing the pacing rate of cardiac cells, however, have recently been developed, including the use of various drugs and pharmacological compositions. Developments in genetic engineering have resulted in methods for genetically modifying cardiac cells to influence their intrinsic pacing rate. For example, U.S. Pat. No. 6,214,620 describes a method for suppressing excitability of ventricular cells by over-expressing (e.g. K+ channels) or under-expressing certain ion channels (e.g. Na+ and Ca2+ channels). PCT Publication No. WO 02/087419 describes methods and systems for modulating electrical behavior of cardiac cells by genetic modification of inwardly rectifying K+ channels (specifically, IK1) in quiescent ventricular cells.
Of particular import to those who suffer from bradyarrhythmias due to insufficient production of If, PCT Publication No. WO 02/098286 describes methods for regulating pacemaker function of cardiac cell via modulation of HCN channels (HCN 1, 2, or 4 isoforms). See also U.S. Patent Application No. 2002/0187948, PCT Application No. WO 02/087419 A2, U.S. Patent Application Publication No. US 2002/0155101A1 and U.S. Pat. No. 6,214,620.
Still, there is a need to improve current methods of using HCN to treat cardiac patients and create pacemaker current capable of being turned on, off and modulated as well as having the capability to react to physiological stimuli to ultimately restore physiological heart rates in patients suffering from arrhythmias.